Dihydrocelastrol inhibits multiple myeloma cell proliferation and promotes apoptosis through ERK1/2 and IL-6/STAT3 pathways in vitro and in vivo

Abstract

Multiple myeloma (MM) is the second most frequent malignant hematological disease. Dihydrocelastrol (DHCE) is synthesized by hydrogenated celastrol, a treterpene isolated from Chinese medicinal plant Tripterygium regelii. In this study, we first reported the anti-tumor activity of DHCE on MM cells. We found that DHCE could inhibit cell proliferation and promote apoptosis through caspase-dependent way in vitro. In addition, DHCE could inactivate the expression of interleukin (IL)-6 and downregulate the phosphorylation of extracellular regulated protein kinases (ERK1/2) and the signal transducer and activator of transcription 3 (STAT3) in MM. It also retained its activity against MM cell lines in the presence of IL-6. Furthermore, treatment of MM cells with DHCE resulted in an accumulation of cells in G₀/G₁ phase of the cell cycle. Notably, DHCE reduced the expression of cyclin D1 and cyclin-dependent kinases 4 and 6 in MM cell lines. Additionally, its efficacy toward the MM cell lines could be enhanced in combination with the histone deacetylase inhibitor panobinostat (LBH589), which implied the possibility of the combination treatment of DHCE and LBH589 as a potential therapeutic strategy in MM. In addition, treatment of NCI-H929 tumor-bearing nude mice with DHCE (10 mg/kg/d, i.p., 1–14 days) resulted in 73% inhibition of the tumor growth in vivo. Taken together, the results of our present study indicated that DHCE could inhibit cellular proliferation and induce cell apoptosis in myeloma cells mediated through different mechanisms, possibly through inhibiting the IL-6/STAT3 and ERK1/2 pathways. And it may provide a new therapeutic option for MM patients.

dihydrocelastrol, multiple myeloma, apoptosis

Introduction

Multiple myeloma (MM) is a fatal malignant B-cell neoplasm characterized by uncontrolled, destructive growth of mutated plasma cells within the bone marrow [1]. MM is the second most prevalent hematological malignancy with a median survival of 3–5 years. Patients over 65 years old are commonly affected by this disease [2]. Current treatment modalities can control the disease for prolonged periods [3]. Recent analyses have shown promising evidence of improved outcomes, likely owing to increasing use of novel treatment agents during initial treatment such as thalidomide and bortezomib [4,5]. However, these drugs have deficiencies that limit their clinical application [6,7], i.e. most myeloma patients are elderly, resistance to novel drugs often appears, and severe side effects, such as peripheral neuropathy and serious infections, occur in many patients [8,9]. Thus, the identification and validation of novel targeted agents to improve clinical outcomes of MM are desirable.

Celastrol, an active compound extracted from the root bark of the Chinese medicine ‘Thunder of God Vine’ (Tripterygium wilfordii Hook F.), was used for years as a natural remedy for inflammatory conditions especially in patients with rheumatoid arthritis, systemic lupus erythematosus, and asthmatics [10–12]. Previous work has also confirmed that celastrol can inhibit cancer cell proliferation and is effective in a wide spectrum of cancers such as prostate cancer [13], leukemia [14], osteosarcoma [15], and human MM [16].

Dihydrocelastrol (DHCE) is a novel dihydroltype analog of celastrol. Westerheide et al. [17] have identified that both DHCE and celastrol have effects on heat shock gene expression. Previous studies have also investigated the functions of DHCE in radiosensitization and SARS-CoV 3CL^pro inhibition [18,19]. However, the related research of DHCE is rare. And to the best of our knowledge, there has been no study on the effects of DHCE upon hematological malignancies. Herein, we explored the anti-tumor activity and mechanisms of DHCE against MM cells both in vitro and in vivo. Our results demonstrated the potential of DHCE to be an effective treatment for MM.

Materials and Methods

Reagents

DHCE was synthesized from celastrol with the method reported by Silverman and his colleagues [20]. The molecular structure is shown in Fig. 1A. DHCE was dissolved in DMSO (Sigma, St Louis, USA) to prepare a 2-mM stock solution and stored at −20°C. Panobinostat and the caspase inhibitor Z-VAD-FMK were purchased from Selleckchem (Houston, USA). Antibodies against caspase-8, caspase-9, caspase-3, poly ADP-ribose polymerase (PARP), ERK1/2, STAT3, β-actin, CDK4, and CDK6 were purchased from Cell Signaling Technology (Beverly, USA); phospho-STAT3 and cyclin D1 were from Abcam (Cambridge, UK). Anti-human Ki-67 protein antibody was from Santa Cruz Biotechnology (Santa Cruz, USA). Human IL-6 was purchased from R&D Systems (Minneapolis, USA). Cell counting kit-8 (CCK-8) was obtained from Dojindo (Dojindo Molecular Technologies Inc., Rockville, USA). Annexin V-FITC and PI detection kit were purchased from Pharmingen (San Diego, USA).

Figure 1.

DHCE induces cytotoxicity in MM cells (A) Chemical structure of DHCE. (B) MM cell lines (ARP-1, OCI-MY5, RPMI-8226, NCI-H929, and NCI-H929R) were treated with DHCE (0.2, 0.4, 0.8, 1.2, and 1.6 μM) for 48 h followed by assessment for cell viability. (C) The viability of ARP-1 and NCI-H929 cells after treatment of DHCE for 24, 48 and 72 h. Both ARP-1 and NCI-H929 cells viability was inversely proportional to the time. The viability showed a decline when the treating time was prolonged. The inhibitory effect had statistical difference compared with control group. (D) Mononuclear cells separated from peripheral blood of three normal donors were treated with DHCE in high doses (3 and 4 μM) for 48 h. Cell viability was then analyzed by CCK-8 assay (P > 0.05). All values are presented as the mean ± SD of three independent experiments performed in duplicate.

MM cell lines and cell culture

Human MM cell lines NCI-H929, ARP-1, OCI-MY5, and RPMI-8226 were purchased from the American Type Culture Collection (ATCC, Manassas, USA). Bortezomib-resistant MM cell line NCI-H929R was kindly provided by Jian Hou (Department of Hematology, Changzheng Hospital, The Second Military Medical University, Shanghai, China). According to Professor Hou's previous report [21], NCI-H929R cell line was obtained by stepwise increasing extracellular concentrations of bortezomib over a period of 8 months. MM cell lines were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS, Sigma), 100 IU/ml penicillin, and 100 μg/ml streptomycin (GIBCO, Grand Island, USA). Cells were maintained at 37°C in 5% CO₂. Culture medium was changed every other day.

Peripheral blood mononuclear cells culture

Peripheral blood mononuclear cells (PBMC) were obtained from normal volunteers in accordance with the Declaration of Helsinki. Approval was obtained from the institutional review board of Shanghai Tenth People's Hospital, Tongji University (Shanghai, China). Mononuclear cells were separated by Ficoll-Hypaque density gradient centrifugation. The purity of PBMC was confirmed by flow-cytometric analysis (BD PharMingen, San Diego, USA). PBMC were cultured in RPMI-IMDM containing 20% FBS, 100 IU/ml penicillin, and 100 μg/ml streptomycin.

Cell proliferation assay

MM cells NCI-H929, RPMI-8226, ARP-1, NCI-H929R, and OCI-MY5 were seeded at a density of 2 × 10⁵ cells per well and treated with increasing concentration of DHCE in a 96-well plate for 24, 48, and 72 h, as previously described [22]. Prior to reading, 8 μl of CCK-8 was added to each well and incubated at 37°C for 2 h. Cells were then measured on a microplate reader (Synergy H4, BioTek, Winooski, USA) at 450 nm to assess proliferation. The half maximal inhibitory concentrations (IC₅₀) of the MM cells in response to treatment were calculated using CalcuSyn software (CalcuSyn; Biosoft, Cambridge, UK).

Cell apoptosis assay

Apoptosis was determined by Annexin V-FITC apoptosis detection kit, as previously described [23]. In our study, apoptotic cells including early (Annexin V positive and PI negative) and late (both Annexin V positive and PI positive) apoptosis.

Cell cycle assay

DHCE-treated and -untreated MM cells were collected, washed with phosphate buffered saline (PBS) and resuspended in 100 μl PBS. Cells were fixed with 70% cold ethanol overnight at −20°C, and then cells were centrifuged at 400 g for 5 min and washed with PBS. Finally, cells were incubated in 500 μl of PI/RNase staining buffer (BD, Franklin Lakes, USA) for 15 min at room temperature in the dark before flow-cytometric analysis.

Western blot analysis

Cells were collected and resuspended in lysis buffer [100 mM Tris-HCl, pH 6.8, 4% sodium dodecyl sulfate (SDS) and 20% glycerol] at 4°C for 30 min. Proteins (30 μg) were fractionated by 10 or 12.5% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto nitrocellulose membrane. The membranes were blocked with 5% skim milk at room temperature for 1 h and incubated with primary antibodies overnight at 4°C, followed by treatment with Fluorescence-conjugated secondary antibodies at room temperature for 1 h. Fluorescence was measured by Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, USA). β-Actin was used to normalize the amount of protein in each sample.

Tumor xenograft models

Male nude mice (5 weeks old) were purchased from Shanghai Laboratory Animal Center (Shanghai, China). Mice were housed in a standard animal laboratory and fed a standard diet with free access to water. NCI-H929 human MM cells (2 × 10⁶) in 100 μl of serum-free culture medium were subcutaneously injected into the right flank region of the nude mice. When tumors were measurable, mice were randomly divided into the control or DHCE group. Mice received daily intraperitoneal injection with 100 μl vehicle (5% DMSO, 15% Tween-80 and saline) or (10 mg/kg DHCE in 5% DMSO, 15% Tween-80 and saline) DHCE. Tumor size and mice weight were assessed each day. Tumor volume was calculated using the following formula: V = length × width²/2. After 14 days of drug administration, the tumors of control group reached around 2500 mm³, all mice were euthanized. All animal studies have been approved by the Animal care and Use Committee of The Tenth People's Hospital of Shanghai, Tongji University. This research was approved by the Science and Technology Commission of Shanghai Municipality.

H&E staining and immunohistochemistry assay

Tumors were dissected and fixed with 4% paraformaldehyde  for 24 h and embedded in paraffin. In addition, 5-μm-thick sections were prepared and stained with H&E. Immunohistochemistry for Ki-67 and cleaved caspase-3 was also performed according to the manufacturer's protocol. In brief, sections were deparaffinized and rehydrated in descending concentrations of alcohol and water. Heat-induced epitope retrieval with EDTA buffer (pH 9.0) for 30 min was followed by endogenous peroxidase blocking with Sniper Blocking Reagent (Biocare, California, USA), and incubation with the primary antibody at 4°C overnight. The primary antibodies used for Ki-67 and cleaved caspase-3 were as described above. Slides were incubated in a prediluted HRP-conjugated secondary antibody. Slides were also conterstained with Mayer's hematoxylin (Sigma Aldrich).

Statistical analysis

All experiments were performed in triplicate. The statistical significance was evaluated with the Student's t-test or one-way variant analysis (ANOVA) by using SPSS 20.0 (SPSS Inc., Chicago, USA). A value of P < 0.05 was considered to be significant. Median dose effect analysis was used to characterize synergistic and antagonistic interactions in conjunction with a commercially available software program (CalcuSyn; Biosoft).

Results

DHCE displays potent cytotoxicity against MM cell lines

DHCE is a novel dihydroltype analog of celastrol with molecular weight of 452.6 and the molecular structure is shown in Fig. 1A. To determine the efficacy of DHCE in MM cell lines, CCK-8 assay in several MM cell lines was first performed to evaluate cell viability. The MM cell lines ARP-1, OCI-MY5, RPMI-8226, NCI-H929, and NCI-H929R were treated with increasing doses of DHCE (0.2, 0.4, 0.8, 1.2, and 1.6 μM) for 48 h. DHCE induced a dose-dependent significant cytotoxicity in all cell lines (Fig. 1B), with the IC₅₀ of ARP-1, OCI-MY5, RPMI-8226, NCI-H929, and NCI-H929R at 0.96 ± 0.06, 0.79 ± 0.04, 0.79 ± 0.08, 0.74 ± 0.03, and 0.81 ± 0.05 μM when treated for 48 h, respectively. ARP-1 and NCI-H929 cells were also treated with DHCE for 24, 48, and 72 h and assessed the cell viability. DHCE decreased cell viability in a time-dependent manner (Fig. 1C). In contrast, DHCE had no cytotoxicity to normal PBMC which were isolated from three normal volunteers. PBMC were treated with DHCE at concentrations as high as 3 and 4 μM. After 48 h of treatment, the cell viability was assessed using CCK-8 assay. No toxicity was found in the PBMC (Fig. 1D) (P > 0.05). These findings indicated that DHCE induces cytotoxicity selectively in MM cells at concentrations that are not cytotoxic to normal cells.

DHCE induces caspase-dependent apoptosis through the extrinsic and intrinsic pathways in MM cell lines

We next examined the cell death triggered by DHCE in ARP-1 and NCI-H929 cells. To quantify the apoptosis, cells treated with DHCE were stained with Annexin-V/PI. The proportion of apoptosis is negligible for control cells, whereas 48 h of exposure to DHCE resulted in a dose-dependent increase of apoptotic cells (Fig. 2A). Interestingly, we also observed higher apoptosis rate at 48 h when compared with that at 24 h (Fig. 2B), which indicated that the effects of DHCE could be enhanced with the time prolonged. These results were consistent with the CCK-8 results. Moreover, DHCE activates apoptosis-related caspase-3, caspase-8, caspase-9, and PARP in ARP-1 and NCI-H929 cells (Fig. 2C), which suggested that the exposure of MM cells to 1 μM DHCE can active both extrinsic and intrinsic pathway of caspase. To further understand the role of apoptosis in the DHCE-induced cell death, we examined cell viability in the presence of a pan-caspase inhibitor, Z-VAD-FMK. As expected, a moderate inhibitory role of Z-VAD-FMK was found in DHCE-induced apoptosis (Fig. 2D). All these data imply that DHCE induces caspase-dependent apoptosis by activating both the extrinsic and intrinsic pathways.

Figure 2.

DHCE induces apoptosis in MM cells (A) Cells were cultured with 0.8, 1.2, and 1.6 μM for 24 and 48 h, stained with Annexin-V/PI and analyzed by flow cytometry. (B) The percentage of FITC-positive cells treated with DHCE. Data are presented as the mean ± SD (n = 3, *P < 0.05). (C) The protein levels of cleaved caspase-3, caspase-8, caspase-9, and PARP were determined by western blot analysis. (D) Both ARP-1 and NCI-H929 cells were pre-incubated with or without Z-VAD-FMK (50 μM) for 3 h, and then treated with DHCE (1 μM) for 48 h, stained with Annexin-V/PI and analyzed by flow cytometry. And the percentage of FITC-positive cells treated with 1 μM of DHCE that pre-incubated with or without Z-VAD-FMK. Data are presented as the mean ± SD (n= 3). *P < 0.05.

DHCE provokes cell-cycle arrest in MM cells

The cell cycle of MM cells treated with DHCE was evaluated by flow cytometry. The percentage of G₀/G₁ phase cells was increased with DHCE treatment at 0.4 μM (46.67% ± 1.92%) and 0.8 μM (60.29% ± 3.01%) compared with the control group (31.12% ± 1.84%) in ARP-1. In NCI-H929 cells, the percentage of G₀/G₁ phase cells was increased with DHCE treatment at 0.4 μM (44.47% ± 1.56%) and 0.8 μM (53.84% ± 2.22%) compared with the control group (36.62% ± 1.27%). (Fig. 3A,B). Western blot analysis was carried out to examine the expression levels of several G₀/G₁-related proteins. The results showed that DHCE downregulated the expression of cyclin D1, CDK4 and CDK6 in ARP-1 and NCI-H929 cells (Fig. 3C). All these data imply that DHCE could provoke cell-cycle arrest in G₀/G₁ phase.

Figure 3.

DHCE induces G₀/G₁phase arrest in MM cells (A) ARP-1 and NCI-H929 cells were treated with DHCE (0, 0.4, and 0.8 μM) for 24 h, stained with PI and analyzed by flow cytometry. (B) Bar graphs show the percentage of cell populations in G₀/G₁, S, or G₂/M phase of cell cycle. Data are presented as the mean ± SD (n = 3). *P < 0.05. (C) The protein levels of cyclin D1, CDK4, and CDK6 were assessed by western blot analysis.

DHCE downregulates the activation of ERK1/2 and IL-6/STAT3 pathways

It has previous been described that the ERK1/2 and IL-6/STAT3 pathways are important for myeloma cell proliferation and survival. Therefore, we assessed the phosphorylated forms of ERK1/2 and STAT3 in MM cells treated with 1 μM DHCE for 48 h. As shown in Fig. 4, DHCE inhibited the phosphorylation of ERK1/2 in both ARP-1 and NCI-H929 cells. However, DHCE had minimal effect on ERK1/2 protein levels. DHCE also reduced the phosphorylation of STAT3 in MM cells without altering the levels of STAT3 protein. IL-6 is a major inducer of STAT3 phosphorylation. We observed that DHCE decreased the IL-6 expression significantly in MM cells (Fig. 4). All these results imply that DHCE could inhibit MM cells through inactivating the ERK1/2 and IL-6/STAT3 pathways.

Figure 4.

Mechanisms of anti-myeloma activity of DHCE (A) ARP-1 and NCI-H929 cells were cultured with 1 μM DHCE for 48 h versus control. Activations of ERK1/2, STAT3 and IL-6 were confirmed using western blot analyses. β-Actin was used as an internal control.

DHCE overcomes the protective effect of IL-6 on MM cells

IL-6 is one of the important growth factors inducing myeloma growth. IL-6 is produced by both autocrine from myeloma cells and paracrine from their microenvironment [24], and IL-6 has the protective effect on MM cells. To test whether DHCE could overcome the protective effect of IL-6 on MM cells, we further carried out the experiment in ARP-1 and NCI-H929 myeloma cells, and found that DHCE still could induce myeloma cell death even in the presence of IL-6 (Fig. 5).

Figure 5.

DHCE overcomes the growth stimulatory effects of IL-6 ARP-1 and NCI-H929 cells were treated with indicated concentrations of DHCE for 48 h in the presence of IL-6 (50 ng/ml). All values are presented as the mean ± SD of three independent experiments performed in duplicate. *P < 0.05 versus control and ^#P > 0.05 versus control.

DHCE inhibits tumor growth in a xenograft model

To examine the in vivo activity of DHCE, we next treated nude mice bearing subcutaneous NCI-H929 cells with either DHCE (10 mg/kg) or a vehicle by intraperitoneal injection daily for 14 days. As shown in Fig. 6A,B, in the model of tumor xenograft of NCI-H929, a significant decrease of tumor volume was observed in DHCE-treated mice compared with the control group (787.82 ± 250.35 mm³ vs. 2655.56 ± 154.49 mm³). Meanwhile, the body weights of the mice in both groups have no significant difference over the course of treatment (Fig. 6C). H&E staining demonstrated the increased necrosis in the tumor of the DHCE-treated group compared with the vehicle-treated control group (Fig. 6D). It was also found that the expression of Ki-67 was low in DHCE-treated mice compared with that in the control group. And DHCE could activate the apoptosis-related caspase-3 in vivo (Fig. 6E,F). These findings indicated that DHCE had anti-tumor effect in vivo. During the experimental period, all mice were monitored to investigate whether DHCE treatment caused lethal toxicity, unhealthy symptoms or gross abnormalities. No evidence of tissue damage or noticeable side effect was observed during visible inspections and microscopic examination of individual organs (data not shown). Consequently, we could suppose that DHCE had anti-tumor effect in MM xenograft model.

Figure 6.

DHCE is active in an MM xenograft model (A) Tumor samples were collected and imaged using a high-definition digital camera. (B) Nude mice bearing subcutaneous NCI-H929 tumors were treated with either DHCE (10 mg/kg; i.p.) or with a vehicle daily for 14 days. Average and standard deviation of tumor volume (cm³) is shown when tumor was measured by caliper (mean tumor volume ± SD, 3 mice/group). (C) The weight of mice was measured daily for 14 days, and data are presented as the mean ± SD. (D) H&E staining tumors from DMSO- or DHCE-treated mice (original magnification: ×400). (E) Ki-67 immunoexpression, ×200. Nuclear Ki-67 immunoexpression levels in DHCE-treated and control groups were significantly different. Ki-67 expression (brown) was weaker in DHCE-treated mice than in the control mice. (F) Cleaved caspase-3 immunoexpression, ×200, showing that the activation of apoptosis-related caspase-3 (brown) was enhanced in the DHCE-treated group.

DHCE synergizes with histone deacetylase inhibitor panobinostat (LBH589) in MM cells

To assess the combined effect of DHCE and LBH589, we evaluated cell proliferation by CCK-8 assay in MM cell line NCI-H929, in the presence of either drug alone or their combination. At the concentration of <32 nM, LBH589 only modestly inhibit the proliferation of NCI-H929 cells. In contrast, cell proliferation was substantially inhibited when LBH589 was combined with low concentrations of DHCE (0.2 or 0.4 μM) (Fig. 7A). The combination index (CI) is considered to be the gold standard to define the synergism of drug–drug interaction [25–27]. CI values = 1 always represent an additive effect, while CI values <1 and >1 mean synergistic and antagonistic interactions, respectively. Thus, a low CI value stands for strong synergism and vice versa. Median dose effect analysis of the interaction between LBH589 (2–32 nM) and DHCE (0.2 or 0.4 μM) yielded CI value <1.0, denoting synergy (Fig. 7B).

Figure 7.

Synergistic interactions between DHCE and LBH589 (A) NCI-H929 cells were treated with LBH589 or LBH589 plus DHCE for 48 h, then assessed for cell viability by CCK-8 assay. (B) CI values were calculated based on median-effect principle. CI values <1.0 denote synergistic interactions. Results are the means of three experiments.

Discussion

DHCE is a dihydroltype analog of celastrol. A previous study has shown that DHCE has effects on heat shock transcription factor 1 (HSF1) similar to celastrol [17]. HSF1 could influence some molecular chaperones, which serve to regulate some heat shock responses [28]. The heat shock responses have been confirmed to be related to a variety of cellular functions, including protein folding, signal transduction, immunity and apoptosis [29–31]. Thus, we inferred that DHCE has a potent anti-tumor effect. However, to date, there has been rare literature in DHCE and its anti-tumor effect on MM. In this study, we investigated the effects of DHCE and found that it inhibited cell proliferation and induced cell death in MM cell lines both in vitro and in vivo.

Apoptosis, as the Type I programmed cell death (PCD), plays a vital role in chemotherapies against a variety of cancers [32]. Chemical compounds that affect apoptotic pathways and eliminate cancer cells are considered promising anti-cancer drugs. In this study, several hallmarks of apoptosis were detected in DHCE-treated MM cells. In the Annexin-V/PI co-staining assay, DHCE-treated cells showed a dose-dependent increase of apoptosis in ARP-1 and NCI-H929 cells (Fig. 2A). And we also observed an obviously higher apoptosis rate at 48 h when compared with that at 24 h (Fig. 2B). Consistent with the CCK-8 results, the effects of DHCE could be enhanced with the time prolonged. Furthermore, we also found that DHCE-triggered apoptosis was associated with the activation of caspase-3, caspase-8, caspase-9, and PARP. And addition of a pan-caspase inhibitor could attenuate DHCE-induced MM cell death. The activation of caspase-8 is linked to the extrinsic apoptotic pathway [33], while the activation of caspase-9 is related to the induction of the intrinsic apoptotic pathway through cytochrome-C release from mitochondria [34]. Therefore, we infer that DHCE may induce cell apoptosis through caspase-dependent pathways.

Cell-cycle checkpoints play important roles in the coordination of cell-cycle transitions in eukaryotic cells and abnormal regulation of cell-cycle checkpoints which frequently occurs in tumor cells [35]. DHCE-induced G₀/G₁ phase arrest is associated with downregulation of cyclin D1, CDK4, and CDK6, which is consistent with previous studies [36–38]. It was reported that inhibition of cyclin D1/CDK4/6 complex activity blocks the progression of cells through G₀/G₁ phase. The Raf/MEK/ERK1/2 and the JAK/STAT3 are survival and proliferative signal pathways and have been shown to be active in MM. Therefore, we examined the effect of DHCE on these cellular signaling pathways. Our results showed that the ERK1/2 and STAT3 were targeted by DHCE. Their phosphorylation was inhibited by DHCE in both NCI-H929 and ARP-1 cells. IL-6 can activate the JAK2/STAT3 and ERK1/2 signal pathways to trigger MM cell proliferation and survival [39–41]. Therefore, the effect of DHCE on IL-6 expression was explored. We found that DHCE could decrease the expression of IL-6 in MM cells. Our data imply that the growth inhibition and apoptosis induced by DHCE in MM cells may be attributed to the abrogation of ERK1/2 and IL-6/STAT3 activation.

We next examined the anti-MM activity of DHCE in an MM xenograft mouse model. Our data showed that treatment with 10 mg/kg DHCE dailyvia the intraperitoneal administration significantly inhibited tumor growth in male nude mice. In addition, no apparent toxicity was observed in DHCE treatment group. These results demonstrated potent in vivo anti-MM activity of DHCE at doses that are well tolerated in mice.

More interestingly, DHCE displays synergism with histone deacetylase (HDAC) inhibitor drugs, panobinostat. Panobinostat, formerly called LBH589, is a novel pan-deacetylase inhibitor that epigenetically modulates classes I, II, and IV HDAC enzymes. In February 2015, FDA granted accelerated approval for the labeling of panobinostat for use in combination with bortezomib and dexamethasone to treat patients with relapsed or refractory MM (RRMM) who have received at least two prior therapies with regimens containing an immunomodulatory drug and bortezomib [42,43]. Therefore, the combination of DHCE with panobinostat may be a promising therapeutic strategy to further improve patient's outcome in MM.

In conclusion, we reported here for the first time the dihydroltype analog of celastrol, DHCE has anti-tumor effects in MM cells via ERK1/2 and STAT3 pathways. Intraperitoneal administration of DHCE is capable of inhibiting tumor growth in the nude mouse xenograft model. Our findings could be able to open new avenues for research toward reducing the threat of MM.

Funding

This work was supported by the grants from the National Natural Science Foundation of China (Nos. 81570190, 81372391, 81529001, and 31271496), and State Key Laboratory of Bioactive Substance and Function of Natural Medicines (No. GTZK201606).

Author notes

†

These authors contributed equally to this work.